Leukocyte stimulating peptides

ABSTRACT

The present application describes peptides that stimulate arachidonic acid release in target cells. The application also discloses peptides that cause intracellular calcium release. The application also discloses methods of using the disclosed peptides.

This application is a divisional of U.S. Ser. No. 10/774,147, filed Feb.5, 2004, which issued as U.S. Pat. No. 7,074,891 on Jul. 11, 2006.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a target cell stimulating peptide. Theinvention is also directed to a method of making the target cellstimulating peptide. Further, the invention is directed to a method ofusing the target cell stimulating peptide as a chemoattractant.

2. General Background and State of the Art

Neutrophils play a key role in innate immune responses. Diverseextracellular agonists modulate neutrophil function by stimulating theactivities of intracellular enzymes (Robson et al. J. Immunol. 2001.167: 1028-1038; M'Rabet et al. J. Biol. Chem. 1999. 274: 21847-21852).Recently, many reports have demonstrated the critical involvement ofphospholipases in neutrophil immune response (Gijon et al. J. Leukoc.Biol. 1999. 65: 330-336; Wu et al. J. Cell Sci. 2000. 113: 2935-2940;Liscovitch et al. Biochem. J. 2000. 345: 401-415). Among thesephospholipases, phospholipase A₂ (PLA₂) is an important enzyme thatmediates several immune responses. PLA₂ hydrolyzes the fatty acyl groupfrom the sn-2 position of phospholipid and concomitantly generateslysophospholipid (Gijon et al. J. Leukoc. Biol. 1999. 65: 330-336; Puriet al. Int. J Biochem. Cell Biol. 1998. 30: 1107-1122). Arachidonic acid(AA), the product of PLA₂ activity, has been implicated in theregulation of various cellular responses, including calcium influx andsuperoxide generation in phagocytic cells (Murthy et al. J. Biol. Chem.1998. 273: 34519-34526; Robinson et al. Biochem. J. 1998. 336: 611-617).

Mammalian cells contain several isozymes of PLA₂, namely, cytosolic PLA₂(cPLA₂), calcium-independent PLA₂, and secretory PLA₂ (Gijon et al. J.Leukoc. Biol. 1999. 65: 330-336; Farooqui et al. J. Neurochem. 1997. 69:889-901). Among the PLA₂ isozymes, cPLA₂ is regarded to play animportant role in agonist-induced AA release and in the regulation oflysophospholipid levels in cells (Gijon et al. J. Leukoc. Biol 1999. 65:330-336). Recently Dana et al. developed cPLA₂-deficient mice andconfirmed the role of cPLA₂ in their eicosanoid production (Dana et al.J. Biol. Chem. 1998. 273: 441-445). Set against this background, cPLA₂is considered to be an important pharmacological target for severalphysiological responses. With this role of PLA₂ in mind, particularlywith respect to neutrophil function, we undertook to identify newligands that modulate PLA₂ activity, and the characterization of theiraction mechanisms.

Several recent studies have reported the use of combinatorial peptidelibraries to identify sequences involved in various biological responses(Boen et al. J. Immunol. 2000. 165: 2040-2047; Wilson et al. J. Immunol.1999. 163: 6424-6434; Hiemstra et al. J. Immunol 1998. 161: 4078-4082).An easy and powerful method for identifying peptide sequences in certainbiological reactions was developed by Houghten et al. (Dooley et al.Methods Mol. Biol. 1998. 87: 13-24). This method, which uses apositional scanning synthetic peptide combinatorial library (PS-SPCL),has been used for various purposes, including the identification ofhuman immunodeficiency virus protease inhibitors, interleukin-8-specificantagonists, the inhibitor for the nuclear factor of activated T cells,and the ligands of opioid receptors, and peptides responsible formodulating leukocytic cell activity (Owens et al. Biochem. Biophys. Res.Commun. 1991. 181: 402-408; Hayashi et al. J. Immunol. 1995. 154:814-824; Aramburu et al. Science. 1999. 285: 2129-2133; Dooley et al. J.Biol. Chem. 1998. 273: 18848-18856; Baek et al. J. Biol. Chem. 1996.271: 8170-8175).

In the present invention, we adopted the PS-SPCL method to identify thepeptides that are responsible for AA release in neutrophil-likedifferentiated HL60 (dHL60) cells. We found 24 peptides that stimulateAA release in dHL60 cells, and found that these peptides act aschemoattractants for human phagocytes. Conversely, on the topic of thereceptors of these peptides, we found that several peptides bound to theformyl peptide receptor like 1 (FPRL1). Some of the peptides were alsofound to bind to other receptor(s) expressed in HL60 cells. In addition,each peptide was found to be capable of stimulating shared and distinctintracellular signaling pathways.

SUMMARY OF THE INVENTION

The invention provides for small polypeptides that induce target cellsto migrate, to release arachidonic acid, induce production ofsuperoxide, or activate PLA₂.

The invention is further directed to the following.

Aspects of the invention include a polypeptide, which is about 4 to 20amino acids in length, and which comprises SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, or SEQ ID NO:35. The polypeptide may be 4 to 15, 4to 10, 4 to 7, or 6 amino acids in length.

Another aspect of the invention includes an antibody that specificallybinds to the polypeptide described above. The antibody may be amonoclonal antibody.

A further aspect of the invention includes an isolated nucleic acidencoding the polypeptide described above. The nucleic acid may includean expression vector comprising the nucleic acid encoding thepolypeptide described above. Another further aspect of the inventionincludes a host cell comprising the expression vector.

Other aspects of the invention include a method of making thepolypeptide described above, comprising (a) synthesizing a polypeptide,which is 4 to 20 amino acids in length; (b) contacting the polypeptidewith a target cell; and (c) determining whether the cells release anarachidonic acid, wherein induction of the arachidonic acid indicatesthe presence of the polypeptide. The target cell may be a leukocyte or aphagocyte.

An additional aspect of the invention includes a method of inducingexpression of arachidonic acid in a target cell, comprising (a)generating a recombinant viral or plasmid vector comprising a DNAsequence encoding the polypeptide described above operably linked to apromoter; and (b) administering the viral or plasmid vector to a patientin need thereof, such that expression of said DNA sequence within thetarget cell results in expression of the arachidonic acid. The targetcell may be a leukocyte or phagocyte.

An additional aspect of the invention includes a method of inducingexpression of arachidonic acid in a target cell comprising contactingthe target cell with the polypeptide described above. The target cellmay be a leukocyte or phagocyte.

An additional aspect of the invention includes a method of activatingPLA₂ in a target cell comprising contacting the cell with thepolypeptide described above. The PLA₂ may be c PLA₂. The target cell maybe a leukocyte or phagocyte.

Another aspect of the invention includes a method of producingsuperoxide in a target cell comprising contacting the cell with thepolypeptide described above. The target cell may be a leukocyte orphagocyte.

An additional aspect of the invention includes a method of causingmovement of a target cell, comprising contacting the cell with thepolypeptide described above. The target cell preferably expresses FPRL1but does not express FPR.

These and other objects of the invention will be more fully understoodfrom the following description of the invention, the referenced drawingsattached hereto and the claims appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description given herein below, and the accompanying drawings,which are given by way of illustration only, and thus are not limitativeof the present invention, and wherein;

FIG. 1 shows an initial screening of the PS-SPCLs for peptidesstimulating AA release in dHL60 cells. Each panel shows the resultsobtained with peptide pools containing known amino acids at each of thesix positions of the hexapeptide. The six positions were individuallydefined (O1, O2 etc.) by one of the 19 L-amino acids. The remaining fivepositions consist of mixtures (X) of the 19 L-amino acids (exceptcysteine). (³H) AA-labeled differentiated HL60 cells (1×10⁶ cells/100μl) were used for each assay. AA release was measured as described inthe Examples. The results are from representative experiments, whichwere conducted in quadruplicate.

FIGS. 2A and 2B show effects of several candidate peptides synthesizedon the basis of the screening results of the PS-SPCLs with respect to AArelease in dHL60 cells. (³H) AA-labeled differentiated HL60 cells werestimulated with 1 μM concentrations of several peptides or 1 μM fMLF,and AA release was measured. The results are presented as means±S.E. ofthree independent experiments. *P<0.01 versus vehicle treatment.Peptides tested include: KKHXXM (SEQ ID NO:37); KKKXXM (SEQ ID NO:38);KKYXXM (SEQ ID NO:39); MKHXXM (SEQ ID NO:40); MKKXXM (SEQ ID NO:41);MKYXXM (SEQ ID NO:42); RKHXXM (SEQ TD NO:43); RKKXXM (SEQ ID NO:44);RKYXXM (SEQ ID NO:45); XKXHKM (SEQ ID NO:46); XKXHPM (SEQ ID NO:47);XKXHRM (SEQ ID NO:48); XKXHVM (SEQ ID NO:49); XKXHYM (SEQ ID NO:50);XKXKKM (SEQ ID NO:51); XKXKPM (SEQ ID NO:52); XKXKRM (SEQ ID NO:53);XKXKVM (SEQ ID NO:54); XKXKYM (SEQ ID NO:55); XKXYKM (SEQ ID NO:56);XKXYPM (SEQ ID NO:57); XKXYRM (SEQ ID NO:58); XKXYVM (SEQ ID NO:59); andXKXYYM (SEQ ID NO:60).

FIG. 3 shows peptide-induced AA release derived from cPLA₂ activation.dHL60 cells were suspended in HBSS containing 0.1% fatty acid-free BSA,incubated for 15 min in the presence or absence of 10 μM of MAFP,AACOCF₃, and BEL at 37° C., and stimulated for 30 min with 1 μM of eachpeptide or vehicle as control. Release of (³H)-arachidonic acid into theextracellular medium was determined with a liquid scintillation counter.Results are expressed as percentages of total cellular radioactivity,mean values±S.E. (n=6) are shown.

FIG. 4 shows effect of PTX on peptide-induced [Ca²⁺]_(i) rise in dHL60cells. dHL60 cells were incubated in the presence or absence of PTX (150ng/ml) for 20 hr and the cells were loaded with fura-2. Thefura-2-loaded dHL60 cells were stimulated with 1 μM of each peptide or500 μM of ATP. The change in 340/380 nm was monitored. Results arerepresentative of 4 independent experiments. Data are presented asmeans±S.E. of four independent experiments.

FIG. 5 shows effect of P24 on [Ca²]_(i) rise in cells of variousorigins. Each cell was loaded with fura-2 for 50 minutes. The cells werestimulated with 10 μM and [Ca²⁺]_(i) increase was monitored. Data arepresented as means±S.E. of three independent experiments.

FIGS. 6A and 6B show chemotactic effect of peptides. Assays wereperformed using a modified Boyden chamber assay, as described in theExamples. Isolated human neutrophils (A) or monocytes (B) (1×10⁶cells/ml in serum free RPMI) were added to the upper wells of a 96-wellchemotaxis chamber and migration across a 3 μm pore size (5 μm formonocytes) polycarbonate membrane was assessed after 2 hrs incubation at37° C. The numbers of migrated cells were determined by counting them ina high power field (400×). Results are presented as means±S.E. of threeindependent experiments each performed in duplicate.

FIG. 7 shows effect of peptides on [Ca²⁺]_(i) rise in FPRL1-expressingRBL-2H3 cells. Fura-2 loaded FPRL1-expressing RBL-2H3 cells werestimulated with 10 μM of each peptide and [Ca²⁺]_(i) increase wasmonitored. The traces shown are from a single experiment representativeof at least three independent experiments.

FIG. 8 shows effect of peptides on [Ca²⁺]_(i) increase in HL60 cells.Fura-2 loaded HL60 cells were stimulated with 10 μM of each peptide and[Ca²⁺]_(i) increase was monitored. The traces shown are from a singleexperiment representative of at least three independent experiments.

FIG. 9 shows regulation of each peptide-stimulated ERK phosphorylationin dHL60 cells. dHL60 cells were preincubated with vehicle or 10 μM ofLY294002, 5 μM of GFX, or 50 μM of PD98059 for 15 min prior to treatmentwith 10 μM of each peptide or vehicle alone for 2 min. Each sample (30μg of protein) was subjected to 8% SDS-PAGE, and phosphorylated ERK wasquantified by immunoblot analysis with anti-phospho-ERK antibody. Theresults shown are from a single experiment representative of at leastthree independent experiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the present application, “a” and “an” are used to refer to bothsingle and a plurality of objects.

As used herein, “about” or “substantially” generally provides a leewayfrom being limited to an exact number. For example, as used in thecontext of the length of a polypeptide sequence, “about” or“substantially” indicates that the polypeptide is not to be limited tothe recited number of amino acids. A few amino acids add to orsubtracted from the N-terminus or C-terminus may be included so long asthe functional activity such as its binding activity is present.

As used herein, “amino acid” and “amino acids” refer to all naturallyoccurring L-α-amino acids. This definition is meant to includenorleucine, ornithine, and homocysteine.

Either single or three letter abbreviations for the amino acids are usedthroughout the application, and may be used interchangeably, and havethe following meaning: A or Ala=alanine; R or Arg=arginine; N orAsn=asparagine; D or Asp=aspartic acid; C or Cys=cysteine; Q orGln=glutamine; E or Glu=glutamic acid; G or Gly=glycine; H orHis=histidine; I or Ile=isoleucine; L or Leu=leucine; K or Lys=lysine; Mor Met=methionine; F or Phe=phenylalanine; P or Pro=proline; S orSer=serine; T or Thr=threonine; W or Trp=tryptophan; Y or Tyr=tyrosine;and V or Val=valine.

As used herein, in general, the term “amino acid sequence variant”refers to molecules with some differences in their amino acid sequencesas compared to a reference (e.g. native sequence) polypeptide. The aminoacid alterations may be substitutions, insertions, deletions or anydesired combinations of such changes in a native amino acid sequence.

Substitutional variants are those that have at least one amino acidresidue in a native sequence removed and a different amino acid insertedin its place at the same position. The substitutions may be single,where only one amino acid in the molecule has been substituted, or theymay be multiple, where two or more amino acids have been substituted inthe same molecule.

Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Also included within the scope of the invention areproteins or fragments or derivatives thereof which exhibit the same orsimilar biological activity and derivatives which are differentiallymodified during or after translation, e.g., by glycosylation,proteolytic cleavage, linkage to an antibody molecule or other cellularligand, and so on.

Insertional variants are those with one or more amino acids insertedimmediately adjacent to an amino acid at a particular position in anative amino acid sequence. Immediately adjacent to an amino acid meansconnected to either the α-carboxy or α-amino functional group of theamino acid.

Deletional variants are those with one or more amino acids in the nativeamino acid sequence removed. Ordinarily, deletional variants will haveone or two amino acids deleted in a particular region of the molecule.

As used herein, “cell stimulating polypeptide” or “cell activatingpolypeptide” refers to a polypeptide that stimulates cells to producearachidonic acid, increase Ca⁺⁺ or act as a chemoattractant.

As used herein, “carriers” include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe pharmaceutically acceptable carrier is an aqueous pH bufferedsolution. Examples of pharmaceutically acceptable carriers includewithout limitation buffers such as phosphate, citrate, and other organicacids; antioxidants including ascorbic acid; low molecular weight (lessthan about 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

As used herein, “chemoattractant” refers to a substance that elicitsdirectional migration of cells in response to the concentration gradientof the molecule.

As used herein, “effective amount” is an amount sufficient to effectbeneficial or desired clinical or biochemical results. An effectiveamount can be administered one or more times. For purposes of thisinvention, an effective amount of a target cell activator compound is anamount that is sufficient to palliate, ameliorate, stabilize, reverse,slow or delay the progression of a condition. In a preferred embodimentof the invention, the “effective amount” is defined as an amount ofcompound capable of stimulating target cells, preferably leukocytes, toproduce arachidonic acid.

As used herein, “host cell” includes an individual cell or cell culturewhich can be or has been a recipient of a vector of this invention. Hostcells include progeny of a single host cell, and the progeny may notnecessarily be completely identical (in morphology or in total DNAcomplement) to the original parent cell due to natural, accidental, ordeliberate mutation and/or change. A host cell includes cellstransfected or infected in vivo with a vector comprising apolynucleotide encoding an angiogenic factor.

As used herein, “leukocyte” refers to a pale, nucleated cell that actsas a part of the immune system by destroying invading cells and removingdebris, and include such cells as granulocyte, lymphocyte, macrophageand monocyte.

Granulocytes or polymorphonuclear leukocytes are marked by the presenceof granules in their cytoplasm, and are active in allergic immunereactions such as arthritic inflammation and rashes. Granulocytesinclude basophils, eosinophils and neutrophils.

Neutrophils move out of blood vessels into infected tissue in order toattack foreign substances such as allergen, bacteria, and so on.Normally, a serious bacterial infection causes the body to produce anincreased number of neutrophils, resulting in a higher than normal whiteblood cell count. Neutrophils perform their function partially throughphagocytosis, a process by which they “cat” other cells and foreignsubstances. For example, the pus in a boil (abscess) is made up mostlyof neutrophils

Lymphocytes are a type of non-granular leukocyte that mainly stays inlymphatic tissue (e.g., the lymph nodes) and is active in immuneresponses, including the production of antibodies.

Macrophage is a type of large leukocyte that travels in the blood butcan leave the bloodstream and enter tissue; like other leukocytes, itprotects the body by digesting debris and foreign cells.

Monocyte is a type of large, round leukocyte that engulfs and breaksdown debris and invading cells. Monocytes are formed in bone marrow andhave round or kidney-shaped nuclei.

As used herein, “ligand” refers to any molecule or agent, or compoundthat specifically binds covalently or transiently to a molecule such asa polypeptide. When used in certain context, ligand may includeantibody. In other context, “ligand” may refer to a molecule sought tobe bound by another molecule with high affinity.

As used herein, “mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep,pigs, and so on. Preferably, the mammal is human.

As used herein, “phagocytes” refer to any cell that engulfs and devoursanother.

As used herein, “purified” or “isolated” molecule refers to biologicalmolecules that are removed from their natural environment and areisolated or separated and are free from other components with which theyare naturally associated.

As used herein, “sample” or “biological sample” is referred to in itsbroadest sense, and includes any biological sample obtained from anindividual, body fluid, cell line, tissue culture, or other source whichmay contain target cells, preferably leukocytes or phagocytes, dependingon the type of assay that is to be performed. As indicated, biologicalsamples include body fluids, such as semen, lymph, sera, plasma, urine,synovial fluid, spinal fluid and so on. Methods for obtaining tissuebiopsies and body fluids from mammals are well known in the art.

As used herein, “sequence identity”, is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in a native polypeptide sequence after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. The % sequence identityvalues are generated by the NCBI BLAST2.0 software as defined byAltschul et al., (1997), “Gapped BLAST and PSI-BLAST: a new generationof protein database search programs”, Nucleic Acids Res., 25:3389-3402.The parameters are set to default values, with the exception of thePenalty for mismatch, which is set to −1.

As used herein, the term “specifically binds” refers to a non-randombinding reaction between two molecules, for example between theinventive polypeptide and the target cells such as leukocytes orphagocytes.

As used herein, “subject” is a vertebrate, preferably a mammal, morepreferably a human.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. “Treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. “Palliating” a disease means that theextent and/or undesirable clinical manifestations of a disease state arelessened and/or the time course of the progression is slowed orlengthened, as compared to a situation without treatment.

As used herein, “vector”, “polynucleotide vector”, “construct” and“polynucleotide construct” are used interchangeably herein. Apolynucleotide vector of this invention may be in any of several forms,including, but not limited to, RNA, DNA, RNA encapsulated in aretroviral coat, DNA encapsulated in an adenovirus coat, DNA packaged inanother viral or viral-like form (such as herpes simplex, andadeno-associated virus (AAV)), DNA encapsulated in liposomes, DNAcomplexed with polylysine, complexed with synthetic polycationicmolecules, complexed with compounds such as polyethylene glycol (PEG) toimmunologically “mask” the molecule and/or increase half-life, orconjugated to a non-viral protein. Preferably, the polynucleotide isDNA. As used herein, “DNA” includes not only bases A, T, C, and G, butalso includes any of their analogs or modified forms of these bases,such as methylated nucleotides, internucleotide modifications such asuncharged linkages and thioates, use of sugar analogs, and modifiedand/or alternative backbone structures, such as polyamides.

Screening for Peptides that Cause Target Cell Stimulation

In the present invention, we screened combinatorial peptide libraries,preferably hexapeptides. We screened a library containing more than 47million different peptide sequences, and identified 24 hexapeptides thatcould stimulate AA release in dHL60 cells. In terms of theirphysiological roles, the peptides were found to enhance superoxidegeneration and the chemotactic migration of phagocytic cells. Throughexperiments on the receptor specificity or the signaling specificity ofthe peptides, we found that the peptides may induce either overlappingor distinct intracellular signals via a common receptor, FPRL1, or viaan unidentified receptor in leukocytic cells.

On investigating the receptor specificity of the peptides, we found that4 peptides could stimulate [Ca²⁺]_(i) increase in FPRL1-expressingRBL-2H3 cells but not in FPR-expressing RBL-2H3 cells (FIG. 7). Of the 4peptides, only two stimulated undifferentiated HL60 cells (FIG. 8).Since undifferentiated HL60 cells do not express FPRL 1, the targetreceptors for these 2 peptides (P18 and P24) could not be FPRL1. Fromexperiments on the effects of PTX on peptide-induced [Ca²⁺]_(i)increase, we found that PTX pretreatment of dHL60 cells completelyinhibited the peptide-induced calcium increase, however, PTX partiallyinhibited the calcium signaling stimulated by P18 or P24 inundifferentiated HL60 cells (data not shown). These results suggest thatthe receptors of peptides in dHL60 cells are coupled to PTX-sensitiveG-proteins, and that the peptide receptors in undifferentiated HL60cells might be coupled to PTX-insensitive G-proteins. These resultsindicate that the receptors of the peptides in undifferentiated HL60cells are not the same as those in dHL60 cells.

Through the study of intracellular signaling pathways by the inventivepeptides, we demonstrated that P14 induced ERK activation via PI3K andPKC, and that P18 induced ERK activation via PKC (FIG. 9). In terms ofthe role of MEK, the 3 peptides, but not P18, caused ERK activation in aMEK-dependent manner (FIG. 9). FIG. 7 shows that 4 peptides stimulated[Ca²⁺]_(i) increase in FPRL1-expressing RBL-2H3 cells. Since dHL60 cellsalso express FPRL1, the 4 peptides may bind to FPRL1 in dHL60 cells.However, the observation that P18 induced ERK is PI3K- orMEK-independent suggests the involvement of another receptor inP18-mediated signaling. We found that P18 and P24 stimulated [Ca²⁺]_(i)increase in undifferentiated HL60 cells (FIG. 8). The results indicatethat P14 and P21 bind to a receptor, such as FPRL 1, and that P18 andP24 bind to at least two receptors, which include FPRL1 in leukocyticcells. In terms of the differential regulation of P18, P24, P14 orP21-induced ERK activation, it can be caused by different spectrum ofreceptors of the peptides.

Although chemoattractants are important immune-modulators and variouschemoattractants (including chemokines) have been identified, applicantshave for the first time identified a few short peptides acting on humanleukocytes. fMLF is a well-known short chemotactic peptide, and has beenuseful for research on phagocyte activation (Pan et al. J. Immunol.2000. 164: 404-411; He et al. J. Immunol. 2000. 165: 4598-4605). Becausethe inventive peptides stimulate human phagocytic cells, such asneutrophils and monocytes, these peptides can also be used as tools forthe study of phagocytic cell functions. In the area of undifferentiatedmyeloma cell activation and signaling, no report has yet been issued onsmall peptides acting on undifferentiated myeloma cells. Because theinventive peptides stimulate undifferentiated HL60 cells, inducing a[Ca²⁺]_(i) increase, they are useful tools for the characterization ofundifferentiated myeloma cell activation.

Peptides that Stimulate Target Cells

In one aspect, the invention is directed to any peptide that is capableof interacting with and activating target cells, preferably leukocytesand phagocytes. In particular, the peptide induces arachidonic acid,induces intracellular release of calcium and induces migration of thetarget cell.

It is understood that the inventive peptides may stimulate or activate atarget cell such as leukocyte or phagocyte by any number of biochemicalor enzymatic mechanisms, so long as the peptide activates the targetcell. Polypeptides that activate target cells include without limitationthe exemplified peptides, which include SEQ ID NO:1 to SEQ ID NO:35.

Nucleic Acid Encoding Polypeptide that Activates Target Cell

By “isolated” polynucleotide sequence, it is intended to encompass anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. This includes segments of DNA encoding the inventivepolypeptide and may further comprise heterologous sequences such asvector sequences or other foreign DNA. For example, recombinant DNAmolecules contained in a vector are considered isolated for the purposesof the present invention, which may be partially or substantiallypurified.

In addition, isolated nucleic acid molecules of the invention includeDNA molecules, which comprise a sequence substantially different fromthose described above but which, either due to the degeneracy of thegenetic code or other variability, still encode the inventivepolypeptide. Thus, it would be routine for one skilled in the art togenerate the variants described above, for instance, to optimize codonexpression or general function for a particular host.

Variant and Mutant Polynucleotides

The present invention further relates to variants of the nucleic acidmolecules which encode proteins, analogs or derivatives of the targetcell activator polypeptide. Such nucleic acid variants include thoseproduced by nucleotide substitutions, deletions, or additions. Thesubstitutions, deletions, or additions may involve one or morenucleotides. Alterations in the amino acid sequence may produceconservative or non-conservative amino acid substitutions, deletions oradditions. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of the polypeptides of the present invention or portionsthereof. Also preferred in this regard are conservative substitutions.

The invention allows for the use of sequences in expression vectors, aswell as to transfect host cells and cell lines, be these prokaryotic oreukaryotic cells. The invention also allows for purification of thepolypeptides expressed from the expression vector. The expression vectormay contain various molecular tags for easy purification. Subsequentlyobtained expression construct may be transformed into any host cell ofchoice. Cell lysates from the host cell is isolated by establishedmethods well known in the field.

Variant and Mutant Polypeptides

To improve or alter the characteristics of the stimulator polypeptide,amino acid engineering may be employed. Recombinant DNA technology knownto those skilled in the art can be used to create novel mutantpolypeptides including single or multiple amino acid substitutions,deletions, additions, or fusion proteins. Such modified polypeptides canshow, e.g., increased/decreased activity or increased/decreasedstability. In addition, they may be purified in higher yields and showbetter solubility than the corresponding natural polypeptide, at leastunder certain purification and storage conditions.

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids which may produce proteins with highlydesirable improved characteristics, such as less aggregation of theproduced polypeptides. Aggregation may not only reduce activity but mayalso be problematic when preparing pharmaceutical formulations, becauseaggregates can be immunogenic.

Antibodies

In one embodiment, the present invention is directed to detecting theactivator polypeptide bound to the target cells using a variety ofdetection methods. One way to detect binding of the activatingpolypeptide to the target cells is to label the activator polypeptidedirectly and assay for its binding using labeling and separationtechniques that are routine to a person of skill in the art. Othermethods include using a labeled ligand that specifically binds to eitherthe activator polypeptide or activator polypeptide/target cell complex.Such a ligand may be an antibody.

Purified activator polypeptide or activator polypeptide/target cellcomplex can be used to produce monoclonal or polyclonal antibody.Subsequently obtained monoclonal or polyclonal antibody can be used todetermine the binding of the activator polypeptide to the target cell invarious samples including cells, tissues, and body fluids such as butnot limited to serum, plasma, and urine. Activator polypeptide oractivator polypeptide/target cell complex may be assayed using a varietyof molecular biological methods, which include but are not limited to insitu hybridization, immunoprecipitation, immunofluorescence staining,Western blot analysis and so on. One can carry out ELISA by usingmonoclonal antibody against activator polypeptide or activatorpolypeptide/target cell complex.

Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. The term“antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Labels

Suitable enzyme labels include, for example, those from the oxidasegroup, which catalyze the production of hydrogen peroxide by reactingwith substrate. Glucose oxidase is particularly preferred as it has goodstability and its substrate (glucose) is readily available. Activity ofan oxidase label may be assayed by measuring the concentration ofhydrogen peroxide formed by the enzyme-labeled antibody/substratereaction. Besides enzymes, other suitable labels include radioisotopes,such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulphur (³⁵S), tritium (³H),indium (¹¹²In), and technetium (^(99m)Tc), and fluorescent labels, suchas fluorescein and rhodamine, and biotin.

Examples of suitable enzyme labels include malate dehydrogenase,δ-5-steroid isomerase, yeast-alcohol dehydrogenase, α-glycerol phosphatedehydrogenase, triose phosphate isomerase, peroxidase, alkalinephosphatase, asparaginase, glucose oxidase, β-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase, and acetylcholine esterase.

Examples of suitable radioisotopic labels include ³H, ¹¹¹In, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci,²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, etc. ¹¹¹In is preferred isotope where in vivoimaging is used since its avoids the problem of dehalogenation of the¹²⁵I or ¹³¹I-labeled polypeptide by the liver. In addition, thisradionucleotide has a more favorable gamma emission energy for imaging.For example, ¹¹¹In coupled to monoclonal antibodies with1-(P-isothiocyanatobenzyl)-DPTA has shown little uptake in non-tumorstissues, particularly the liver, and therefore enhances specificity oftumor localization.

Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd,⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, and ⁵⁶Fe.

Examples of suitable fluorescent labels include an ¹⁵²Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoerythrin label, a phycocyanin label, an allophycocyanin label, ano-phthaldehyde label, and a fluorescamine label.

Examples of suitable toxin labels include, Pseudomonas toxin, diphtheriatoxin, ricin, and cholera toxin.

Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, and an aequorin label.

Examples of nuclear magnetic resonance contrasting agents include heavymetal nuclei such as Gd, Mn, and iron. Deuterium may also be used. Othercontrasting agents also exist for EPR, PET or other imaging mechanisms,which are known to persons of skill in the art.

Typical techniques for binding the above-described labels topolypeptides are provided by Kennedy et al. (1976) Clin. Chim. Acta70:1-31, and Schurs et al. (1977) Clin. Chim. Acta 81:1-40. Couplingtechniques include the glutaraldehyde method, the periodate method, thedimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide estermethod, all of which methods are incorporated by reference herein.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingthe inventive polypeptide are administered to activate the target cell,such as leukocyte or phagocyte to enhance its immune response tobacteria or any other foreign substance by way of gene therapy. Genetherapy refers to therapy performed by the administration to a subjectof an expressed or expressible nucleic acid. In this embodiment of theinvention, the nucleic acids produce their encoded protein that mediatesa therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

Therapeutic Composition

In one embodiment, the present invention relates to treatment forvarious conditions that are characterized by lack of sufficient targetcell activation. In this way, the inventive therapeutic compound may beadministered to human patients who are either suffering from, or proneto suffer from the disease or condition by providing compounds thatstimulate target cell activation. Preferably, the target cell may beleukocyte or phagocyte. In particular, the disease or condition may beassociated with infection by various infectious pathogens such asviruses or bacteria. Further in particular, the present invention isdirected to treatment for an infectious disease accompanied byattenuation of normal immune response, such as acquired immunedeficiency syndrome or cancer.

The formulation of therapeutic compounds is generally known in the artand reference can conveniently be made to Remington's PharmaceuticalSciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. For example,from about 0.05 μg to about 20 mg per kilogram of body weight per daymay be administered. Dosage regime may be adjusted to provide theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. The activecompound may be administered in a convenient manner such as by the oral,intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (eg using slowrelease molecules by the intraperitoneal route or by using cells e.g.monocytes or dendrite cells sensitized in vitro and adoptivelytransferred to the recipient). Depending on the route of administration,the peptide may be required to be coated in a material to protect itfrom the action of enzymes, acids and other natural conditions which mayinactivate said ingredients.

For example, the low lipophilicity of the peptides will allow them to bedestroyed in the gastrointestinal tract by enzymes capable of cleavingpeptide bonds and in the stomach by acid hydrolysis. In order toadminister peptides by other than parenteral administration, they willbe coated by, or administered with, a material to prevent itsinactivation. For example, peptides may be administered in an adjuvant,co-administered with enzyme inhibitors or in liposomes. Adjuvantscontemplated herein include resorcinols, non-ionic surfactants such aspolyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzymeinhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DEP) and trasylol. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes.

The active compounds may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propylene glycoland liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsuperfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, chlorobutanol, phenol, sorbic acid, theomersal and the like. Inmany cases, it will be preferable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecomposition of agents delaying absorption, for example, aluminiummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterile active ingredient into a sterile vehicle which containsthe basic dispersion medium and the required other ingredients fromthose enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze-drying technique whichyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

When the peptides are suitably protected as described above, the activecompound may be orally administered, for example, with an inert diluentor with an assimilable edible carrier, or it may be enclosed in hard orsoft shell gelatin capsule, or it may be compressed into tablets, or itmay be incorporated directly with the food of the diet. For oraltherapeutic administration, the active compound may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 1% by weightof active compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 5 toabout 80% of the weight of the unit. The amount of active compound insuch therapeutically useful compositions is such that a suitable dosagewill be obtained. Preferred compositions or preparations according tothe present invention are prepared so that an oral dosage unit formcontains between about 0.1 μg and 2000 mg of active compound.

The tablets, pills, capsules and the like may also contain thefollowing: A binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound may be incorporated intosustained-release preparations and formulations.

As used herein “pharmaceutically acceptable carrier and/or diluent”includes any and all solvents, dispersion media, coatings antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, use thereofin the therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth is impaired.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form. A unit dosageform can, for example, contain the principal active compound in amountsranging from 0.5 μg to about 2000 mg. Expressed in proportions, theactive compound is generally present in from about 0.5 μg/ml of carrier.In the case of compositions containing supplementary active ingredients,the dosages are determined by reference to the usual dose and manner ofadministration of the said ingredients.

Delivery Systems

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis, construction of a nucleicacid as part of a retroviral or other vector, etc. Methods ofintroduction include but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody or a peptide of the invention, care must be taken to usematerials to which the protein does not absorb. In another embodiment,the compound or composition can be delivered in a vesicle, in particulara liposome. In yet another embodiment, the compound or composition canbe delivered in a controlled release system. In one embodiment, a pumpmay be used. In another embodiment, polymeric materials can be used. Inyet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose.

A composition is said to be “pharmacologically or physiologicallyacceptable” if its administration can be tolerated by a recipient animaland is otherwise suitable for administration to that animal. Such anagent is said to be administered in a “therapeutically effective amount”if the amount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient patient.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims. The following examples are offered by way ofillustration of the present invention, and not by way of limitation.

EXAMPLES Example 1 Materials and Methods

Fmoc amino acids were obtained from Millipore (Bedford, Mass.),Rapidamide resin from Dupont (Boston, Mass.), peripheral bloodmononuclear cell (PBMC) separation medium (Histopaque-1077), cytochromec, and fMLF from Sigma (St. Louis, Mo.), fura-2 pentaacetoxymethylester(fura-2/AM) from Molecular Probes (Eugene, Oreg.), RPMI 1640 from LifeTechnologies (Grand Island, N.Y.), dialyzed fetal bovine serum andsupplemented bovine serum from Hyclone Laboratories Inc. (Logen, Utah),pertussis toxin (PTX), GF109203X, and PD98059 from Calbiochem (SanDiego, Calif.), and LY294002 was purchased from BIOMOL ResearchLaboratories, Inc. (Polymouth Meeting, Pa.).

Example 2 Cell Culture and HL60 Cell Differentiation

U937 (human histiocytic lymphoma cells), HL60 (human promyelocyticleukemia cells), Raw 264.7 (mouse macrophage), Jurkat (human acute Tcell leukemia), PC12 (rat adrenal pheochromocytoma cells), 3Y1 (Ratembryonic fibroblasts), 3T3L1 (preadipocytes), and NCI-H292 (humanmucoepidermoid pulmonary carcinoma cells) were obtained from theAmerican Type Culture Collection (Rockville, Md.) and maintained asrecommended. FPR- or FPRL1 expressing RBL-2H3 cells were cultured asdescribed previously (He et al. J. Immunol. 2000. 165: 4598-4605). Cellswere maintained at about 1×10⁶ cells/ml under standard incubatorconditions (humidified atmosphere, 95% air, 5% CO₂, at 37° C.). HL60cells were induced to differentiate into the granulocyte phenotype byadding dimethylsulfoxide (DMSO) (final concentration 1.25%, v/v) for 4days to the culture medium, as described previously (Itoh et al. Blood.1998. 92: 1432-1441).

Example 3 Isolation of Leukocytes

Peripheral blood leukocyte concentrates were donated by the Ulsan RedCross Blood Center (Ulsan, Korea). PBMCs were separated on aHistopaque-1077 gradient. After two washings with HBSS without Ca²⁺ andMg²⁺, the PBMCs were suspended in 10% FBS containing RPMI and incubatedfor 60 min at 37° C. to let the monocytes attach to the culture dish.Cells were washed 5 times with warmed RPMI medium to remove lymphocytes,and then the attached monocytes were collected, as described previously(Bae et al. J. Leukoc. Biol. 1999. 65: 241-248). Human neutrophils wereisolated according to standard procedures; i.e., dextran sedimentation,hypotonic lysis of erythrocytes, and using a medium lymphocyteseparation gradient, as described previously (Seo et al. J. Immunol.1997. 158: 1895-1901). Isolated human leukocytes were then usedpromptly.

Example 4 Preparation of Peptide Libraries, and the Synthesis andAnalysis of Peptides

The hexapeptide libraries were prepared in the Peptide Library SupportFacility of Pohang University of Science and Technology, as describedpreviously (Baek et al., J. Biol. Chem. 1996. 271: 8170-8175,incorporated by reference herein in its entirety). Finally, 114 peptidepools (Cys was excluded from the library constructions) wereindividually dissolved in water to a final concentration of 27 nM perpeptide. The peptides were synthesized by the solid-phase methoddescribed previously (Baek et al. J. Biol. Chem. 1996. 271: 8170-8175).Briefly, peptides were synthesized on a Rapidamide support resin andassembled following the standard Fmoc/t-butyl strategy on an acid-labilelinker. The composition of peptides was confirmed by amino acidanalysis, as described previously (Baek et al. J. Biol. Chem. 1996. 271:8170-8175).

Example 5 Initial Screening of the PS-SPCLs and the Measurement of AARelease

For the initial screening of the PS-SPCLs, we measured the AA releasestimulating activity of each peptide pool. Cultured dHL60 cells (10⁷cells/ml) were pre-labeled with 0.5 μCi/ml of (³H)-AA in RPMI 1640medium containing 10% FBS for 90 min at 37° C. in a humidified incubatorsupplied with 95% air and 5% CO₂, as described previously (Bae et al. J.Immunol. 2000. 164: 4089-4096). The labeled cells were then washed twicewith serum-free RPMI 1640 and incubated in RPMI 1640 medium containing0.1% fatty acid-free BSA for 15 min at 37° C. After discarding themedium, the cells were stimulated with various concentrations of peptidefor the indicated times. Radioactivity in the medium and of collectedcells was determined with a liquid scintillation counter. Wheninvestigating the effects of inhibitors, cells were preincubated withthe indicated concentrations of each inhibitor or vehicle for 15 minprior to stimulation.

Example 6 Measurement of [Ca²⁺]_(i)

The level of [Ca²⁺]_(i) was determined using Grynkiewicz's method usingfura-2/AM (Grynkiewicz et al. J. Biol. Chem. 1985. 260: 3440-3550).Briefly, prepared cells were incubated with 3 μM of fura-2/AM at 37° C.for 50 min in serum-free RPMI 1640 medium under continuous stirring.2×10⁶ cells were aliquoted for each assay in Ca²⁺ free Locke's solution(154 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl₂, 5 mM HEPES, pH 7.3, 10 mMglucose, and 0.2 mM EGTA). Fluorescence changes were measured at thedual excitation wavelength of 340 nm and 380 nm, and the calculatedfluorescence ratio was translated into [Ca²⁺]_(i).

Example 7 Measurement of Superoxide Generation

We determined superoxide anion generation by measuring cytochrome creduction using a microtiter 96 well plate ELISA reader(Bio-Tekinstruments, EL312e, Winooski, Vt.) as described previously (Baeet al. Blood. 2001. 97: 2854-2862). Human neutrophils (1×10⁶ cells/100μl of RPMI 1640 medium per well of a 96-well plate) were preincubatedwith 50 μM of cytochrome c at 37° C. for 1 min and then incubated withthe indicated peptide concentrations. Superoxide generation wasdetermined from change in light absorption at 550 nm over 5 minutes at 1min intervals.

Example 8 Chemotaxis Assay

Chemotaxis assays were performed using multiwell chambers (NeuroprobeInc., Gaithersburg, Md.) (Bae et al. Blood. 2001. 97: 2854-2862).Briefly, prepared human monocytes were suspended in RPMI at aconcentration of 1×10⁶ cells/ml, and 25 μl of the suspension was thenplaced onto the upper well of a chamber separated by a 5 μmpolyhydrocarbon filter (3 μm pores size not polyvinylpyrrolidone coated,as is needed for neutrophils) from peptides orN-formyl-methionyl-leucyl-phenylalanine (fMLF) in the lower well. Afterincubation for 2 hours (90 minutes for neutrophils) at 37° C.,non-migrated cells were removed by scraping, and cells that migratedacross the filter were dehydrated, fixed, and stained with hematoxylin(Sigma, St. Louis, Mo.). Stained cells were counted in five randomlychosen high power fields (HPF) (400×) (Bae et al. Blood. 2001. 97:2854-2862).

Example 9 Results Example 9.1 Identification of Peptides that StimulateAA Release in dHL60 Cells

We screened 114 peptide pools (around 47 million different peptides)from hexapeptide PS-SPCLs to identify those peptides that stimulate AArelease in dHL60 cells. FIG. 1 shows the results of the initialscreening. Amino acids in different positions of the hexapeptidesinduced different levels of AA release stimulating activity. The mostactive peptide/position combinations were peptides having the formulaXKXXXM (SEQ ID NO:1), wherein Lys (K), Met (M), or Arg (R) are in thefirst position, Lys (K) in second, His (H), Lys (K), or Tyr (Y) inthird, His (H), Lys (K), or Tyr (Y) in fourth, Lys (K), Pro (P), Arg(R), Val (V), or Tyr (Y) in fifth, and Met (M) in sixth.

Based on the results of the first screening of the peptide libraries, wegenerated by reiterative synthesis peptide pools containing1×1×1×3×5×1=15 or 3×1×3×1×1×1×=9 individual hexapeptides. We then testedthe effectiveness of these peptide pools for AA release stimulatingactivity in dHL60 cells using the same methods as used in the initialscreening (FIGS. 2A and 2B). After this second screening, we found thatKKHXXX (SEQ ID NO:2), KKYXXX(SEQ ID NO:3), RKYXXX (SEQ ID NO:4), MKYXXX(SEQ ID NO:5), XXXHKM (SEQ ID NO:6), XXXHVM (SEQ ID NO:7), XXXYKM (SEQID NO:8), XXXYPM (SEQ ID NO:9), XXXYVM (SEQ ID NO:10), or XXXYYM (SEQ IDNO:11) were most active (FIGS. 2A and 2B). Finally, 24 differentsynthesized peptides are listed in Table I and measured for their effecton AA release in dHL60 cells. All of these 24 peptides stimulated AArelease at a concentration of 10 μM (Table I), and (K/R/M)KYY(P/V/Y)M(P10, P11, P12, P16, P17, P18, P22, P23, and P24), (R/M)KYHVM (P14, P20)and MKYYKM (P21) were the most potent (Table I).

TABLE I Effect of inventive peptides on arachidonic acid release indifferentiated HL60 cells^(a) Folds of increase Peptide Sequence (% oftotal) P1 KKHHKM-NH₂ 1.25 ± 0.168 (SEQ ID NO: 12) P2 KKHHVM-NH₂ 1.23 ±0.153 (SEQ ID NO: 13) P3 KKHYKM-NH₂ 1.45 ± 0.306 (SEQ ID NO: 14) P4KKHYPM-NH₂ 1.41 ± 0.247 (SEQ ID NO: 15) P5 KKHYVM-NH₂ 1.68 ± 0.390 (SEQID NO: 16) P6 KKHYYM-NH₂ 1.52 ± 0.296 (SEQ ID NO: 17) P7 KKYHKM-NH₂ 1.42± 0.226 (SEQ ID NO: 18) P8 KKYHVM-NH₂ 1.30 ± 0.170 (SEQ ID NO: 19) P9KKYYKM-NH₂ 1.49 ± 0.268 (SEQ ID NO: 20) P10 KKYYPM-NH₂ 2.27 ± 0.199 (SEQID NO: 21) P11 KKYYVM-NH₂ 2.49 ± 0.023 (SEQ ID NO: 22) P12 KKYYYM-NH₂2.58 ± 0.168 (SEQ ID NO: 23) P13 RKYHKM-NH₂ 1.47 ± 0.220 (SEQ ID NO: 24)P14 RKYHVM-NH₂ 2.23 ± 0.403 (SEQ ID NO: 25) P15 RKYYKM-NH₂ 1.71 ± 0.214(SEQ ID NO: 26) P16 RKYYPM-NH₂ 2.57 ± 0.450 (SEQ ID NO: 27) P17RKYYVM-NH₂ 2.82 ± 0.210 (SEQ ID NO: 28) P18 RKYYYM-NH₂ 2.61 ± 0.295 (SEQID NO: 29) P19 MKYHKM-NH₂ 1.68 ± 0.221 (SEQ ID NO: 30) P20 MKYHVM-NH₂2.55 ± 0.271 (SEQ ID NO: 31) P21 MKYYKM-NH₂ 2.86 ± 0.426 (SEQ ID NO: 32)P22 MKYYPM-NH₂ 2.95 ± 0.668 (SEQ ID NO: 33) P23 MKYYVM-NH₂ 3.05 ± 0.401(SEQ ID NO: 34) P24 MKYYYM-NH₂ 2.93 ± 0.323 (SEQ ID NO: 35)^(a)Arachidonic acid release was measured in [³H] arachidonicacid-labeled cells stimulated with 10 μM of peptide.

Example 9.2 Effect of Isozyme-Specific Inhibitors of PLA₂ on thePeptide-Stimulated AA Release

To address the question as to which isoform of PLA₂ is responsible forpeptide-induced AA release, several isoform-specific inhibitors of PLA₂were added together with several representative peptides, and were foundto stimulate AA release at 1 μM in dHL60 cells (FIG. 3). Pretreatment ofthese cells with the cPLA₂-specific inhibitors, AACOCF₃ and MAFP blockedthe induction of AA by 4 of the peptides, P14, P18, P21, and P24 (FIG.3). 10 μM of MAFP or AACOCF₃ almost completely prevented AA release asinduced by the 4 peptides, whereas another PLA₂ inhibitor, BEL, known tobe specific for iPLA₂, did not interfere with peptide-induced AA release(FIG. 3). AA stimulated release by these peptides was also inhibited bythe chelation of intracellular Ca²⁺ with BAPTA/AM, which also supportsthe notion of cPLA₂ activation (data not shown). These results,therefore, indicate that the 4 peptides evoke AA release by stimulatingcPLA₂ but not iPLA₂ in dHL60 cells.

Example 9.3 Effect of Peptides on [Ca²⁺]_(i) Rise in dHL60 Cells

It is well known that intracellular calcium elevation is essentiallyrequired for the activation of cPLA₂ (Gijon et al. J. Leukoc. Biol.1999. 65: 330-336). The finding that peptide-stimulated AA release isinhibited by preincubating dHL60 cells with a cPLA₂ inhibitor, MAFP, ledus to investigate whether the peptides affect [Ca²⁺]_(i) increase. Asshown in Table II, many of the peptides caused an increase in [Ca²⁺]_(i)after stimulation at 1 μM in dHL60 cells, though some peptides, like P1and P7 did not affect [Ca²⁺]_(i) increase (Table II). The concentrationdependency of the peptide-induced [Ca²⁺]_(i) increase was alsoinvestigated. P3, P4, P5, and P6 showed maximal activity atconcentrations exceeding 20 μM (data not shown), P10, P11, P12, P16,P17, P18, P22, P23, and P24 showed maximal activity at approximately 3μM (data not shown). A number of reports have demonstrated that manyextracellular ligands modulate cellular activities via PTX-sensitiveG-protein(s) in human leukocytic cells (Sano et al. J. Immunol. 2000.165: 2156-2164; Badolato et al. J. Immunol. 1995. 155: 4004-4010). Toinvestigate the possible involvement of PTX-sensitive G-protein inpeptide-induced [Ca²⁺]_(i) increases, dHL60 cells were treated with PTX(150 ng/ml) for 20 hr prior to the addition of each of the 24 peptides.As shown in FIG. 4, each active peptide-induced [Ca²⁺]_(i) rise wasalmost completely inhibited by PTX. ATP, a ligand that does not act onPTX-sensitive G-protein-coupled receptors, stimulated [Ca²⁺]_(i)increases in dHL60 cells and this [Ca²⁺]_(i) rise was not inhibited byPTX (FIG. 4). These results indicate that the peptides stimulate[Ca²⁺]_(i) release via PTX-sensitive G-protein in dHL60 cells.

TABLE II Effect of inventive peptides on intracellular calcium increasein differentiated HL60 cells^(b) Peptide [Ca²⁺]_(i) (nM) P1  0 P2  0 P3  8 ± 3.2 P4  14 ± 4.1 P5  34 ± 9.5 P6   50 ± 11.7 P7  0 P8  31 ± 3.5 P9  85 ± 15.8 P10 152 ± 28.7 P11 158 ± 25.3 P12 153 ± 13.2 P13 10 ± 4.2 P1485 ± 9.5 P15  77 ± 12.1 P16 135 ± 21.4 P17 143 ± 10.2 P18 150 ± 14.5 P1913 ± 3.0 P20 155 ± 16.3 P21 122 ± 15.7 P22 168 ± 21.6 P23 153 ± 13.2 P24165 ± 23.4 ^(b)Intracellular calcium increase was monitored in fura-2loaded cells stimulated with 1 μM of peptide.

Example 9.4 Cell Type Specificity of the Peptides

Since the synthesized peptides stimulated neutrophil-like dHL60 cells,we checked their effects on neutrophils, a type of leukocyte. Thestimulation of neutrophils with one of the peptides, P24, resulted in a[Ca²⁺]_(i) rise (FIG. 5). Monocytes and U937 cells were also activatedby P24 (FIG. 5), but Raw 264.7 and Jurkat cells were not activated byP24 (FIG. 5). Next, we examined the effects of P24 on [Ca²⁺]_(i) rise inseveral non-leukocytic cell lines. However, 3Y1, PC12, NCI—H292, andHUVEC cell lines showed no response to P24 in terms of [Ca²⁺]_(i) rise(FIG. 5 and data not shown). These results indicate that the peptideeffects are neutrophil and monocyte specific. The other active peptidesshowed similar results in terms of their leukocyte-specificities (datanot shown).

Example 9.5 Effect of the Peptides on Superoxide Generation

Superoxide generation is one of the important steps in the host'sdefense mechanism by phagocytes (Lambeth et al. J. Bioenerg. Biomembr.1988. 20: 709-733). We tested the effect of the 4 representativepeptides (P14, P17, P21, and P24) on superoxide generation in humanneutrophils. These 4 peptides were found to stimulate superoxidegeneration in a concentration-dependent manner in human neutrophils(data not shown). Moreover, the stimulation of human neutrophils with 1μM of each peptide caused a dramatic change in superoxide generation(Table III). P24 was the most potent in terms of superoxide generationin human neutrophils (Table III).

TABLE III Effect of peptides on superoxide generation in humanneutrophils^(c) Peptide Superoxide production (nmole/10⁶ cells) P14  9.3± 1.42 P18 28.4 ± 3.21 P21 12.7 ± 1.76 P24 34.2 ± 0.48 fMLF 26.5 ± 1.32^(c)Superoxide production was measured by monitoring the amount ofcytochrome c reduction caused by stimulating with 1 μM of peptide.

Example 9.6 Chemotactic Effect of Peptides on Leukocytes

We found that 4 peptides (P14, P18, P21, and P24) stimulated superoxidegeneration and [Ca²⁺]_(i) increase in human phagocytic cells. Thesepeptide-induced phagocyte activation phenomena are similar tochemoattractant-induced phenomena. Therefore, we checked whether thepeptides exhibited chemotactic activity on human monocytes orneutrophils. The 4 active peptides induced migration of humanneutrophils over a concentration range of 1-10 μM (FIG. 6A). The maximalcellular migration-inducing activity mediated by the peptides was morethan 200% of that induced by 1 μM of fMLF (FIG. 6A). The 4 peptides(P14, P17, P21, and P24) also induced cellular chemotaxis in humanmonocytes (FIG. 6B). Moreover, the 4 peptides caused monocyte chemotaxisin a concentration range of 0.01 to 10 μM (FIG. 6B). An inactive controlpeptide, LFMYHP (SEQ ID NO:36), did not induce cellular chemotaxis inneutrophils or monocytes at concentrations less than 10 μM (FIGS. 6A and6B). In four experiments with independently prepared leukocytes, the 4peptides showed similar cellular migration-inducing activity.

Example 9.7 Receptor Specificity of the Peptides: Effect on FPRL1

Peptide induced phagocyte activation was found to be very similar tothat induced by chemoattractants. Formyl peptide receptor, FPR, andFPRL1 are well-known chemoattractant receptors in neutrophils (Le et al.Immunol. Rev. 2000. 177: 185-194; Le et al. Cytokine Growth Factor Rev.2001. 12: 91-105). To examine whether the peptides bind to FPR or FPRL1we investigated the effect of the peptides on [Ca²⁺]_(i) increase inFPR- or FPRL1-expressing RBL-2H3 cells. No peptide was found to affect[Ca²⁺]_(i) in FPR-expressing RBL-2H3 cells (data not shown). However,several peptides including 4 peptides (P14, P18, P21, and P24) inducedcalcium increase in FPRL1-expressing RBL-2H3 cells (FIG. 7 and data notshown). An inactive control peptide (LFMYHP (SEQ ID NO:36)) was foundnot to be able to induce calcium increase in FPRL1 cells (FIG. 7). Amongthe active peptides, the potency of calcium increasing activities wasfound to be different for each peptide. These results indicate thatseveral peptides, including the 4 peptides (P14, P18, P21, and P24) areligands for FPRL1 but not for FPR.

Example 9.8 Differentiation Status Specificity of the 4 Peptides in HL60Cells

FIG. 5 shows that the peptides acted on leukocytic cells but not onnon-leukocytic cells. Many extracellular ligands have been reported tohave cellular differentiation status specificity (Rabin et al. J.Immunol. 1999. 162: 3840-3850; Berardi et al. Blood. 1995. 86:2123-2129). We investigated whether the peptides showed suchdifferentiation status specificity in myelocytes, by checking the effectof these peptides on [Ca ²⁺]_(i) increase in undifferentiated anddifferentiated HL60 cells. As shown in Table II, the 4 peptidesstimulated [Ca²⁺]_(i) increase in dHL60 cells. When undifferentiatedHL60 cells were stimulated with the 4 peptides, [Ca²+]_(i) was found tobe dramatically induced by P18 and P24 (FIG. 8). The other 2 peptides,P14 and P21, did not affect [Ca²⁺]_(i) increase in HL60 cells (FIG. 8).Unlike neutrophils or dHL60 cells, undifferentiated HL60 cells do notexpress FPR or FPRL1 on the cell surface (Prossnitz et al. J. Immunol.1993. 151: 5704-5715). We also confirmed that fMLF (a FPR-specificligand) or lipoxin A4 (a FPRL1-specific ligand) did not affect[Ca²⁺]_(i) increase in HL60 cells, indicating that HL60 cells do notexpress FPR or FPRL1. These results suggest that receptors other thanFPRL1 are activated by the peptides P18 and P24. Moreover, P14 and P21stimulated dHL60 cells and FPRL1-expressing RBL-2H3 cells, demonstratingthat 2 peptides show differentiation status specificity.

Example 9.9 Comparison of Intracellular Signaling by the 4 Peptides

Extracellular signal regulated protein kinase (ERK) is a well-knownintracellular enzyme that mediates diverse cellular responses (Sugden etal. Cell Signal. 1997. 9: 337-351). Many reports have demonstrated thatchemoattractants stimulate ERK activity, and that this may result inseveral pivotal stages in the modulation of leukocytic cells (Woo et al.J. Biol. Chem. 2002. 277: 8572-8578; Brill et al. J. Immunol. 2001. 166:7121-7127). In the present study, we found that the stimulation of dHL60cells with 4 peptides (P14, P18, P21, and P24) caused a dramaticincrease in the phosphorylation level of ERK (FIG. 9). Moreover, thesepeptide-induced ERK activation was time-dependent, and showed maximalactivity 5 minutes after stimulation (data not shown). To compare theintracellular signaling involving these 4 peptides, dHL60 cells werepretreated either with LY294002 (50 μM), GF109203X (5 μM), or PD98059(50 μM) or left untreated as a control. After being incubated for theindicated periods (15 minutes for LY294002 and GF109203X, 60 minutes forPD98059), the cells were stimulated with 1 μM of each peptide for 5minutes. As shown in FIG. 9, P14-induced ERK phosphorylation was blockedby LY294002, GF109203X, or PD98059, indicating that the peptide-inducedERK activation is phosphatidylinositol-3-kinase (PI-3K), protein kinaseC (PKC), or MEK-dependent. P18-induced ERK phosphorylation wascompletely blocked by GF109203X but not by LY294002 (FIG. 9). PD98059partially blocked P18-induced ERK phosphorylation (FIG. 9). P21 alsocaused ERK phosphorylation showing PI3K and MEK-dependency (FIG. 9), andP24-induced ERK phosphorylation was partially blocked by LY294002 butnot by GF109203X (FIG. 9). These results suggest that the 4 peptidesstimulate overlapping and non-overlapping intracellular signalingpathways, which result in the activation of ERK in dHL60 cells.

All of the references cited herein are incorporated by reference intheir entirety.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention specifically described herein. Suchequivalents are intended to be encompassed in the scope of the claims.

1. A polypeptide, which is 6 to about 20 amino acids in length, andwhich consists of or comprises the peptide of SEQ ID NO:29.
 2. Thepolypeptide according to claim 1, which is 6 to about 15 amino acidslong.
 3. The polypeptide according to claim 2, which is 6 to about 10amino acids long.
 4. The polypeptide according to claim 3, which is 6 toabout 7 amino acids long.
 5. The polypeptide according to claim 4, whichis 6 amino acids long.
 6. A method of inducing production of arachidonicacid in a target leukocyte or phagocyte cell comprising contacting thetarget cell with the polypeptide of claim
 1. 7. A method of activatingcPLA₂ in a target leukocyte or phagocyte cell comprising contacting thetarget cell with the polypeptide according to claim 1, wherein saidactivating of cPLA₂ is detected by measuring production of arachidonicacid.
 8. A method of producing superoxide in a target leukocyte orphagocyte cell comprising contacting the cell with the polypeptideaccording to claim
 1. 9. A method of causing movement of a culturedtarget cell, comprising contacting the cultured target cell with thepolypeptide according to claim 1.